The urokinase-type plasminogen activator receptor (uPAR) has been found to be overexpressed on the surface of cancer cells, as well as their associated neovasculature, in virtually every cancer type tested, including primary, metastatic, and chemotherapy-resistant cancers. uPAR is an important master regulator that drives extracellular matrix degradation and angiogenesis, and cancer cell proliferation, adhesion, and migration. These processes in turn allow rapid tumor growth and metastasis to other sites in the body. Clinical studies indicate overexpression of uPAR and its soluble ligand uPA are independent predictors of low disease- free and overall survival. Thus, uPAR is an extremely attractive target for therapeutic intervention; however, the absence of an FDA-approved drug that inhibits uPAR highlights a critical need for new approaches to effectively block the activity f this receptor. We will apply our expertise in molecular and cellular biology, biochemistry, and protein engineering to develop a novel first-in-class biologic capable of effectively blocking uPAR-mediated cancer growth, metastasis, and tumor-associated angiogenesis. In Aim 1, a bispecific inhibitor will be created by fusing two natural protein ligands that bind to distinct epitopes on uPAR and modulate different biological activities. This bispecific protein will be further engineered using combinatorial methods to create an ultra-high affinity uPAR inhibitor that will: 1) simultaneously bind two uPAR epitopes with high affinity and specificity, 2) prevent uPAR from associating with its two main ligands and a multitude of other cell-surface receptors, and 3) inhibit multiple signaling pathways involved in cancer growth, metastasis, and tumor-associated angiogenesis. In Aim 2, engineered bispecific proteins will be compared to monospecific proteins for their ability to inhibit uPAR-mediated cell signaling, as well as tumor cell proliferation, adhesion, migration, and survival. Numerous uPAR antagonists have been developed over the last two decades, including small molecules, peptides, protein ligands, and antibodies. However, the efficacies of these inhibitors have been limited owing to their low affinity relative to the native uPAR ligands, and/or their inability to simultaneously block all uPR functions that drive cancer growth and metastasis. Protein engineering, combined with multispecific target binding, will generate in uPAR inhibitors with orders of magnitude higher binding affinity compared to previously developed antagonists. The resulting biologics have exciting potential to overcome critical barriers that have prevent development of successful uPAR-targeted therapeutics, and would represent a potential breakthrough for targeted therapy of aggressive cancers.